Semiconductor nanocrystals, often referred to as quantum dots (QDs), offer a viable alternative to presently used isotopic and non-isotopic detection systems for use in biomolecular research protocols and assays, as well as for clinical and diagnostic assays. The goal of these systems is the detection and reporting of a specific molecule that is indicative of the presence of a certain molecular, cellular or organismal entity, or of the occurrence of a particular molecular event, such as the transcription of a particular gene or the production of a particular protein within an organism. While isotopic detection systems offer a high degree of sensitivity, there are inherent problems associated with their use such as safety and disposal concerns, half-life of the isotope and where very low levels of the target molecule are present, the length of film exposure time (e.g. up to 7 days) required in order to detect a signal. While non-isotopic systems offer a safety advantage, the fluorescent reporter molecules are often susceptible to rapid fading (i.e. photobleaching). As well, while many of the currently available non-isotopic detection systems are highly sensitive, these systems rely upon the use of a secondary-antibody detection regimen wherein the actual detection is of a fluorescent or chromatogenic agent linked to a secondary-antibody targeted against a primary-antibody that binds to an antigen linked to a molecular probe against the target molecule. Successful utilization of such systems requires the use of expensive reagents that often exhibit only a limited storage or shelf-life, and further requires a user to perform a number of procedural steps, the less-than-optimal performance of which may lead to a false-negative result.
Interest from the medical and research communities regarding quantum dots stems largely from the unique optical and electrical properties that are associated with QDs. In comparison to organic fluorophores, certain types of QDs possess up to twenty times greater luminescence, are highly resistant to photobleaching, exhibit narrow spectral linewidths, and are size and materials-tuneable so as to be excitable using only a single wavelength. Problematic, however, is the fact that in order for QDs to be used in the context of a biological setting, for example, imaging and detection of and within live cells, the QD must possess a coating that makes the QD bio-compatible with biological systems, such as being aqueously soluble, and at the same time does not lessen the stability of the QD under physiological conditions. Overcoming this problem is exasperated by the fact that QDs are generally synthesized in an organic solvent as the hydrophobic solvent ligands act as stabilizing agents for QD nucleation and growth, and inhibit the aggregation of the QDs during their synthesis.
In terms of their basic structure, the synthesis of a QD comprising an inner nanoparticle-sized semiconductor “core” together with an outer semiconductor “cap” that is of a different material than the core and which binds to the core is a process that is well known in the art (U.S. Pat. Nos. 6,468,808 and 6,699,723). Usually, the QD core is selected from a combination of Group IIB–VIB, Group IIIB–VB or Group IVB—IVB elements from the periodic table, while the cap is selected from a material that, in combination with the core, results in a luminescent quantum dot. The cap is selected to passivate the core by having a higher band gap than the core, and as such, the cap is preferred to be a semi-conducting material from the Group IIB–VIB combination of elements from the periodic table.
The luminescent properties of QDs result from quantum size confinement, which occurs when metal and semiconductor core particles are smaller than their exciton Bohr radii, about 1 to 5 nm (Alivisatos, Science, 271, 933–37 (1996); Alivisatos, J. Phys. Chem., 100, 13226–39 (1996); Brus, Appl. Phys., A 53, 465–74 (1991). It is known that an improvement in the QD luminescence results from the capping of a size-tunable lower band gap core particle with a higher band gap shell. For example, CdSe quantum dots passivated with a ZnS layer are strongly luminescent (35 to 50% quantum yield (QY)) at room temperature, and their emission wavelength can be tuned from blue to red by changing the particle size. Moreover, the ZnS capping protects the core surface and leads to greater stability of the quantum dot (Hines et al., J. Phys. Chem., 100, 468–471 (1996); and Dabbousi et al., J. Phys. Chem. B 101, 9463–75 (1997)). Despite having these greater luminescent capacities, such capped QDs are not water-soluble and are thus not suitable for use in biological systems.
To date, numerous attempts have been made to produce a QD that has a bio-compatible surface that does not promote non-specific binding of the QD to molecules, does not cause an abatement of the optical properties of the QD, nor increase the size of the QD, nor negate the ability of the QD to be further coated with a desired molecule(s) of choice, but allows for the large-scale and cost effective production of the QD. QDs have been provided that have their surface modified through the addition of amphiphilic polymers, phospholipids, dendrimers, oligomeric ligands, biofunctional molecules such as deoxyribonucleic acid (DNA), and genetically-modified proteins (Chan and Nei, Science, 281, 2016–2018 (1998); Bruchez et al., Science, 281, 2013–2016 (1998); Mattoussi et al., J. Am. Chem. Soc., 125, 12142–12150 (2000); Kim and Bawendi, J. Am. Chem. Soc., 125, 14652–14653 (2003); Dubertret et al., Science, 298, 1759–1762 (2002); Wang et al., J. Am. Chem. Soc., 124, 2293 (2002); Wu et al., Nature Biotechnology, 21, 41–46 (2003); Guo et al., J. Am. Chem. Soc., 125, 3901 (2003)). While such modifications impart water solubility to the QD, such surface modifications do not allow cost-effective, commercial scale production. In an effort to provide a thin, secure organic shell around a QD without increasing the diameter of the QD so as to render the QD inaccessible to target systems or limit the number of QDs that can be attached to a target, Kim and Bawendi (J. Am. Chem. Soc., 125, 14652–14653 (2003)) have succeeded in surrounding QDs with an oligomeric phosphine shell. Problematic, however, is that the approach put forward by Kim and Chan requires the complex synthesis of a stabilizing and interfacing oligophosphine ligand, thereby severely limiting the potential for the large scale production of such QDs.
It would be thus advantageous to provide a QD that has a coating that would allow for the QD to be used in conjunction with biological systems. Any coating that is provided should allow for the maintenance of long-term monodispersity of the QDs in an aqueous environment, not promote non-specific binding of the QD to other molecules, not detract from the optical properties of the QD when compared to the organic solvent soluble counterpart of the coated QD, maintain the small size of the QD, allow for the QD to be further coated with biomolecules of a range of types, and allow for the QD to be produced on a commercial scale in a cost-effective manner.